In complex environments such as cheeses, the lack of relevant information on the physiology and virulence expression of pathogenic bacteria and the impact of endogenous microbiota has hindered progress in risk assessment and control. Here, we investigated the behaviour of Staphylococcus aureus, a major foodborne pathogen, in a cheese matrix, either alone or in the presence of Lactococcus lactis, as a dominant species of cheese ecosystems. The dynamics of S. aureus was explored in situ by coupling a microbiological and, for the first time, a transcriptomic approach. Lactococcus lactis affected the carbohydrate and nitrogen metabolisms and the stress response of S. aureus by acidifying, proteolysing and decreasing the redox potential of the cheese matrix. Enterotoxin expression was positively or negatively modulated by both L. lactis and the cheese matrix itself, depending on the enterotoxin type. Among the main enterotoxins involved in staphylococcal food poisoning, sea expression was slightly favoured in the presence of L. lactis, whereas a strong repression of sec4 was observed in cheese matrix, even in the absence of L. lactis, and correlated with a reduced saeRS expression. Remarkably, the agr system was downregulated by the presence of L. lactis, in part because of the decrease in pH. This study highlights the intimate link between environment, metabolism and virulence, as illustrated by the influence of the cheese matrix context, including the presence of L. lactis, on two major virulence regulators, the agr system and saeRS.